1. Field of the Invention
The invention relates to a method of producing a target protein as soluble protein.
2. Related Art
A large number of recombinant protein expression systems have been developed to date, including, for example, cell-free translation systems and recombinant protein expression systems within hosts such as bacteria, yeast, insects, transgenic animals, and transgenic plants. Escherichia coli is widely used as an expression system for heterologous protein because it is easily grown to high densities and because of the progress in research on host vector systems.
However, when a target protein is expressed using these recombinant protein expression systems, incorrect folding by the expressed protein can prevent expression of the functionality of the original protein and can result in the not insignificant formation of insoluble aggregates, known as inclusion bodies. Even when, for example, refolding is carried out in such cases after solubilization of the inclusion body with a denaturant or surfactant, the correctly folded protein exhibiting its native functionality is not necessarily obtained. In addition, even when protein expressing its original functionality is obtained, in many instances a satisfactory recovery rate is not obtained.
Against this background, a method of suppressing the formation of inclusion bodies of an expressed recombinant target protein has not been established to date. As a stand in for such a method, expression as a soluble protein is attempted by fusing the insoluble target protein with the soluble high molecular weight (40,000) maltose-binding protein or glutathione S-transferase (GST) (Fox, J. D. and Waugh, D. S., “Maltose-binding protein as a solubility enhancer.” METHODS MOL. BIOL., 205: 99-117 (2003); Ausubel, F. M. et al., editors, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Vol. 2, 16.0.1 (1996)). However, there have been problems such as, for example, the soluble protein may not exhibit its original activity or functionality and the target protein may become insoluble when the maltose-binding protein or GST is removed.
The ZZ domain is a synthetic IgG binding region developed on the basis of the IgG binding region of protein A (refer, for example, to Nilsson B. et al., Protein Eng., 1: 107-113 (1987)).
However, in those cases where the ZZ domain of the IgG binding region has been expressed fused with a target protein, there have been no reports of an effect whereby the solubility of the fusion protein is increased, nor have there been reports of an activity that contributes to an efficient refolding of the target protein to the active form of the protein. Up to the present time, the use of the ZZ domain originating from protein A has not gone beyond use, after expression of the fusion protein with a target protein, as a ligand in IgG antibody affinity chromatography in target protein purification. In addition, IgG antibody columns are expensive and there are only limited instances where they can be used even when a genetic recombinant fusion protein utilizing the ZZ domain is employed in mass production.